*Using the correct template mass and primer concentrations is crucial. If you submit less than the recommended ng of DNA, sequencing fails more often.

+

* This is the standard UW price, but volume discounts are possible if our lab starts using more.

-

*If you have a low concentration plasmid prep, you can increase the mass you send by sending 0.5 uL of 100 uM primers (the stock concentration) instead of 5 uL of 5 uM primers.

+

-

==Understanding Results==

+

== SUBMIT TO UW By REQUEST Box A ==

-

*[[User:Janet B. Matsen|Janet]] recommends doing a multiple alignment in NCBI's Blast webpage. Do not use the APE program's aligner, especially if you have a repetitive sequence.

+

* In summer of 2013 we got a GeneWiz box outside our lab. To use this box, select "UW Box A," the by-request box, so they know to pick up our samples. They don't come up to our box if nobody submitted a sample.

-

**If you get an imperfect match or fail to match to your expected sequence, you can loosen the stringency requirements by selecting "somewhat dissimilar" or "more dissimilar" instead of "highly similar."

+

-

**Always have good labels in your FASTA files to make sure you don't mix things up.

+

==Preparing Samples==

-

**If you are making variants of a construct, you can compare your sequencing results to all of the things you are making. First make a text document with each of the reference sequences you are creating by pasting them back to back in a text editor. Include descriptions for each e.g. "> SH3-FLS in pSB1A3". Paste your sequence from GeneWiz into the top box and all of the other sequences in the bottom box. When the results are returned, click the "max score" button & it will put the match with the highest score first. This is most likely your product, but a sequencing read can match to multiple designs depending on your project.

+

*15 uL total including 5 uL of 5 uM primers.

+

**Keep a fresh stock of 5 uM primers that you take good care of (keep clean) and replace often with fresh stock.

**Low concentration minipreps (<70 ng/uL) frequently fail when submitted to sequencing. It may be due to impurities that co-elute; if you submit many uL of this to meet the 500 ng/sample requirement, then you will have more of these impurities present. -[[User:Janet B. Matsen|Janet 3/20/2012]]

==When Sequencing Fails==

==When Sequencing Fails==

-

*There are several ways it can fail & you can see the GeneWiz help for causes/solutions below.

+

*There are several ways it can fail & you can see the GeneWiz help for causes/solutions below. You can also watch this [http://www.genewiz.com/file/dnaseq201/dnaseq201.html GeneWiz webinar]. Don't hesitate to call GeneWiz customer support -- their customer service is incredible.

*GeneWiz has very high standards for passing sequencing reactions. About 1/2 of your attempts may fail. Sometimes you can look at the trace file and decide it is good enough on your own despite a failed label.

**multiple are present on your DNA or you accidentally have a mixture of DNA templates.

+

***You can prepare a new miniprep, with the hope that your first one was contaminated with another plasmid. Otherwise, you have to assume the plasmid you built accidentally contains an extra priming site. Of course, miniprepping from single colonies will reduce the possibility of mixed templates.

+

***Note: a plasmid can contain an extra priming site via pcr/assembly fluke OR the insert you add can contain a mispriming site.

**carry-over inhibitors in the reaction such as phenol, ethanol, EDTA, or salts

+

+

===[https://clims3.genewiz.com/Customer/Support/Troubleshooting/Early%20Termination.pdf Early termination]===

+

*hairpin present, repetitive sequence, or high GC content can terminate the rxn sequencing. Note: they have several "difficult sequencing" protocols that can be used; select them in the form.

+

*too much DNA

+

**results in smears. They can dilute the sample you sent and re-run it. Use 10ng/uL/kb of DNA. [[image:GeneWiz - Too Much DNA.png|thumb]]

+

*bubble in capillary

+

**can appear as a smear. You won't know when this happens, but customer support may be able to tell.

+

*dye blobs [[image:dye_blob_GeneWiz.png|thumb]]

+

**too many terminating nucleotides remain after their purification -- see image on right. A big peak covers the sequence you want. They generally have it at the beginning, so you should start your priming ~ 100 bp away from the sequence of interest. They happen on PCR products and plasmid & are best visually edited (ignored.)

+

+

===[https://clims3.genewiz.com/Customer/Support/Troubleshooting/High%20Background.pdf High Background]===

+

* what is the difference between High Background & non-specific?

+

** Customer support says: the degree to which the background sequence

+

** you can trust a sequence that fails because of high background more than you can non-specific

+

+

=== When it fails, try again! ===

*You can submit 2 samples per order for a free repeat, but they must be on the same templates you submitted.

*You can submit 2 samples per order for a free repeat, but they must be on the same templates you submitted.

-

*You can resequence as many as you like for 1/2 price and you have the choice to use new preps. This allows you to use a fresh miniprep or primer batch. If you want to submit fresh samples for 1/2 price, put in a new order and note which are free repeats, and their respective order number in the "Notes" section of the form.

+

*You can resequence as many as you like for 1/2 price and you have the choice to use new preps. This allows you to use a fresh miniprep or primer batch. If you want to submit fresh samples for 1/2 price, you should contact customer service and get approval. Write in the comments section at the bottom which samples are 1/2 price repeats, and which row & order the original failed sequence was in. Then call them with the order number for this new request/order. [[User:Janet B. Matsen|Janet 7/18/2012]]

*Whether or not your repeat will be successful depends on several variables such as:

*Whether or not your repeat will be successful depends on several variables such as:

**the concentration and quality of the sample -- if a "no priming" or "poor quality" samples is "no priming" or "poor quality" after you sequence again, the quality and quantity might be too bad to work the 2nd time.

**the concentration and quality of the sample -- if a "no priming" or "poor quality" samples is "no priming" or "poor quality" after you sequence again, the quality and quantity might be too bad to work the 2nd time.

**you also may have a GC-rich region, hairpin or general difficult template that is causing binding issues, a problem with the binding site, a problem with the primer, etc.

**you also may have a GC-rich region, hairpin or general difficult template that is causing binding issues, a problem with the binding site, a problem with the primer, etc.

+

+

== Vague primer design instructions ==

+

*"We recommend designing your sequencing primers in a region that is 100 bases upstream of your sequence of interest. If you do not have the luxury of having this buffer, the closest you want the primer to be to your area of interest is 50-60 bases. Anything closer and you risk missing a portion of your area of interest. The primers should be about 18-24 bases in length with a Tm of 56-60 degrees. The GC content should be about 45-55%. Many vendors that provide oligo synthesis services have software into which you can plug your primer sequence to check for Tm, GC content and homodimerization. For the oligo purity, desalting is all that is needed for sequencing" ([http://www.genewiz.com/public/DNA-sequencing-FAQs.aspx link])

+

** Note that Finnzymes reports Tm higher than other calculators, so 62-65oC is probably good if using a Finnzyme calculation.

+

+

== Sequencing gel purified PCR products ==

+

Organic solvents inhibit/inactivate the Taq used in Sanger sequencing.

+

=== Tip sent by technical support to Janet 5/2013: ===

+

* Tips for Column Purification of Template DNA

+

** Here are a few guidelines to help aid in the effective column purification of your product.

+

**This will help to eliminate carry over of ethanol into the eluate.

+

# Prior to elution, there is a wash step that includes a high concentration of ethanol. If you are using a micro spin column, after the wash step, please spin samples at maximum speed in the microfuge 2 times for 2 minutes decanting the flow through completely after the first spin and placing the column in a fresh tube for elution after the second spin.

+

# If there is a rim around the membrane, carefully remove residual ethanol using a pipette tip after the second spin and then let the column sit for 2 minutes on the bench top prior to elution in order to promote residual ethanol evaporation.

+

# Carefully elute with EB or water heated to 50'C by adding drop wise to the membrane surface.

SUBMIT TO UW By REQUEST Box A

In summer of 2013 we got a GeneWiz box outside our lab. To use this box, select "UW Box A," the by-request box, so they know to pick up our samples. They don't come up to our box if nobody submitted a sample.

Preparing Samples

15 uL total including 5 uL of 5 uM primers.

Keep a fresh stock of 5 uM primers that you take good care of (keep clean) and replace often with fresh stock.

GeneWiz sample submission guideline

Red Flags:

Low concentration minipreps (<70 ng/uL) frequently fail when submitted to sequencing. It may be due to impurities that co-elute; if you submit many uL of this to meet the 500 ng/sample requirement, then you will have more of these impurities present. -Janet 3/20/2012

When Sequencing Fails

There are several ways it can fail & you can see the GeneWiz help for causes/solutions below. You can also watch this GeneWiz webinar. Don't hesitate to call GeneWiz customer support -- their customer service is incredible.

GeneWiz has very high standards for passing sequencing reactions. About 1/2 of your attempts may fail. Sometimes you can look at the trace file and decide it is good enough on your own despite a failed label.

multiple are present on your DNA or you accidentally have a mixture of DNA templates.

You can prepare a new miniprep, with the hope that your first one was contaminated with another plasmid. Otherwise, you have to assume the plasmid you built accidentally contains an extra priming site. Of course, miniprepping from single colonies will reduce the possibility of mixed templates.

Note: a plasmid can contain an extra priming site via pcr/assembly fluke OR the insert you add can contain a mispriming site.

hairpin present, repetitive sequence, or high GC content can terminate the rxn sequencing. Note: they have several "difficult sequencing" protocols that can be used; select them in the form.

too much DNA

results in smears. They can dilute the sample you sent and re-run it. Use 10ng/uL/kb of DNA.

bubble in capillary

can appear as a smear. You won't know when this happens, but customer support may be able to tell.

dye blobs

too many terminating nucleotides remain after their purification -- see image on right. A big peak covers the sequence you want. They generally have it at the beginning, so you should start your priming ~ 100 bp away from the sequence of interest. They happen on PCR products and plasmid & are best visually edited (ignored.)

you can trust a sequence that fails because of high background more than you can non-specific

When it fails, try again!

You can submit 2 samples per order for a free repeat, but they must be on the same templates you submitted.

You can resequence as many as you like for 1/2 price and you have the choice to use new preps. This allows you to use a fresh miniprep or primer batch. If you want to submit fresh samples for 1/2 price, you should contact customer service and get approval. Write in the comments section at the bottom which samples are 1/2 price repeats, and which row & order the original failed sequence was in. Then call them with the order number for this new request/order. Janet 7/18/2012

Whether or not your repeat will be successful depends on several variables such as:

the concentration and quality of the sample -- if a "no priming" or "poor quality" samples is "no priming" or "poor quality" after you sequence again, the quality and quantity might be too bad to work the 2nd time.

you also may have a GC-rich region, hairpin or general difficult template that is causing binding issues, a problem with the binding site, a problem with the primer, etc.

Vague primer design instructions

"We recommend designing your sequencing primers in a region that is 100 bases upstream of your sequence of interest. If you do not have the luxury of having this buffer, the closest you want the primer to be to your area of interest is 50-60 bases. Anything closer and you risk missing a portion of your area of interest. The primers should be about 18-24 bases in length with a Tm of 56-60 degrees. The GC content should be about 45-55%. Many vendors that provide oligo synthesis services have software into which you can plug your primer sequence to check for Tm, GC content and homodimerization. For the oligo purity, desalting is all that is needed for sequencing" (link)

Note that Finnzymes reports Tm higher than other calculators, so 62-65oC is probably good if using a Finnzyme calculation.

Sequencing gel purified PCR products

Organic solvents inhibit/inactivate the Taq used in Sanger sequencing.

Tip sent by technical support to Janet 5/2013:

Tips for Column Purification of Template DNA

Here are a few guidelines to help aid in the effective column purification of your product.

This will help to eliminate carry over of ethanol into the eluate.

Prior to elution, there is a wash step that includes a high concentration of ethanol. If you are using a micro spin column, after the wash step, please spin samples at maximum speed in the microfuge 2 times for 2 minutes decanting the flow through completely after the first spin and placing the column in a fresh tube for elution after the second spin.

If there is a rim around the membrane, carefully remove residual ethanol using a pipette tip after the second spin and then let the column sit for 2 minutes on the bench top prior to elution in order to promote residual ethanol evaporation.

Carefully elute with EB or water heated to 50'C by adding drop wise to the membrane surface.